Inmunologically active proteins on a T-cell: T-cell receptor, CD-4, CD-28, PD-1 and CTLA-4 and a calcium channel
Immunologically active proteins on a T-cell. TCR (blue), CD-4 (light blue), CD-28 (dark blue), PD-1 (magenta), CTLA-4 (violet), Ca-channel (dark violet). The T-cell receptor, CD-4 and CD-28 activate T-cells, while PD-1 and CTLA-4 inhibit the activation of T-cells. [selvanegra/GEtty Images]

Scientists at Gladstone Institutes, UC San Francisco (UCSF), and Stanford School of Medicine report they have released the most detailed map to date of how complex networks of genes work together and the resultant effects these networks have on immune cell function and on the development of immune disease. The researchers say this new information on immune networks can aid in both the development of new immunotherapies as well as further our understanding of autoimmune diseases.

“These results help us flesh out a systematic network map that can serve as an instruction manual for how human immune cells function and how we can engineer them for our benefit,” said Alex Marson, MD, PhD, director of the Gladstone-UCSF Institute of Genomic Immunology and co-senior author of the study.

The research, conducted in collaboration with Jonathan Pritchard, PhD, professor of genetics and of biology at Stanford School of Medicine, was published Monday in Nature Genetics. 

Using CRISPR-Cas9

Researchers have been working in this area for some time—in which the levels of proteins in immune cells change and interact with one another—and represent these gene-protein connections as a network that can look like a subway map. Developing these network maps is important as they can reveal why mutations in two genes can lead to the same disease, or how a single drug may have an impact on several immune proteins at once.

But the new research differs from other approaches taken in the past which knocked out a single gene for each protein to see how that single gene-protein combination impacts other genes and proteins and the overall function of the immune cell. According to the researchers in this new work, such a “downstream” approach only reveals half of what happens in the cells.

“We really wanted to look at what is controlling key immune genes,” said Jacob Freimer, PhD, a postdoctoral fellow in both the Marson and Pritchard labs, and the paper’s first author. “This kind of upstream approach hadn’t been done before in primary human cells.”

Using the subway map metaphor, this “upstream” approach would map the routes by first identifying the route’s main hubs and then figuring out the routes taken to reach these key areas, rather than identifying the satellite stations and then trying to reconstruction the network using that information.

To do this, the team turned to CRISPR-Cas9 to perturb thousands of genes at once and focusing on genes that make transcription factors—the switches that turn genes on and off and often control many genes at once. Taking this approach the investigators studied how disruption of transcription factors on three genes—IL2RA, IL-2, and CTLA4—that are known to play an important role in T cell function were impacted. These three genes were the hubs the researchers focused on for their upstream mapping of the networks.

Highly connected networks

While the researchers anticipated they would discover connections between genes regulating IL2RA, IL-2, and CTLA, they didn’t expected to find such a high level of connectivity. Of the 117 regulators found to control levels of at least one of the three genes, 39 controlled two of the three, and 10 regulators simultaneously altered levels of all three genes. The team then switched to the more traditional downstream approach by removing 24 of the pinpointed regulators from T cells to show the full list of genes they regulate aside from IL2RA, IL-2, and CTLA4.

This allowed the researchers to show that many of the regulators controlled each other. In one instance the transcription factor IRF4 altered the activity of nine other regulators and was itself regulated by 15 other regulators all 24  of which controlled levels of IL2RA. In other cases, regulators were themselves regulated by IL2RA, in so-called “feedback loops.”

“There were cases where a transcription factor was regulating IL2RA, but then IIL2RA itself also controlled that same transcription factor,” said Freimer. “It appears that these kinds of feedback loops and regulatory networks are much more interconnected than we previously realized.”

Clinical implications

The new details of the map could have important implications for the treatment and management of a number of immune diseases as the team found a high number of genes already linked to multiple sclerosis, lupus, and rheumatoid arthritis.

The study suggests there’s a central network of key genes, and when this network is perturbed, it can increase a person’s risk of developing disease. It also suggests there is a core group of genes that could be targeted by drug to treat immune diseases.

“When we understand the ways in which these networks and pathways are connected, it starts to help us understand key collections of genes that need to function properly to prevent diseases of the immune system,” Marson concluded.

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